The long term goal of this study is to define new therapeutic targets of osteoclast related bone diseases, such as osteoporosis and tumor bone metastasis. The immediate goal of this study is to understand the mechanisms underlying the transcription factors that regulate osteoclast lineage commitment and differentiation. The critical need for the treatments of osteoclast related bone diseases has been exacerbated by the recent discovery that the negative effects (e.g., jaw osteonecrosis) of currently used osteoclast inhibitors may outweigh the benefits. The mechanism underlying transcription factors that specify osteoclast lineage commitment, differentiation, and function remains unclear. It is also necessary to clarify why M-CSF alone induces precursors to differentiate into macrophages, while both M-CSF and RANKL induce precursors to differentiate into osteoclasts. Further study is needed to address these biological questions. As a step towards understanding the transcription factors which modulate osteoclast gene (e.g., cathepsin K) expression and osteoclast differentiation, we are characterizing the cathepsin K critical cis-regulatory elements (CCREs). We found a CCAAT/enhancer-binding protein ? (C/EBP?) binding site as a CCRE and confirmed C/EB? as its trans-regulatory factor as the CCRE binding protein (CCREBP). Our Preliminary Studies further demonstrated that only the combined stimulation of M-CSF and RANKL will induce C/EBP? high expression; that in vitro C/EBP? knockdown blocks osteoclast differentiation and overexpression increase osteoclast formation; and that C/EBP? knockout in mice (C/EBP?-/-) impaired osteoclastogenesis and resulted in an osteopetrotic phenotype. Based on our Preliminary Study data, we hypothesize that C/EBP? is the key osteoclastogenesis transcription factor that regulates osteoclast lineage commitment and differentiation in the M-CSF and RANKL-dependent signaling pathway(s) through interactions with its heterodimerization partners and regulation of osteoclast genes. We will test this hypothesis through four specific aims: in Aim 1, we will investigate the role of C/EBP? in osteoclast differentiation by characterizing the phenotypes and pathomechanism of C/EBP? null mutation mice (C/EBP?-/-) compared with C/EBP?+/+ mice. In Aim 2, we will reveal the function of C/EBP? at various stages of osteoclast differentiation through characterization of the phenotypes and pathomechanism of three C/EBP? conditional knockout mouse models. In Aim 3, we will define in vitro the roles of C/EBP? at different stages of osteoclast differentiation using RNAi knockdown as a loss-of-function study and retrovirus overexpression as a gain-of-function study. In Aim 4, we dissect the mechanism of C/EBP? interactions that confers osteoclastic specificity by characterizing C/EBP? heterodimerization partners, downstream genes and their functions. This study will not only improve our understanding of the role of transcription factors in normal osteoclast differentiation and function and in osteolytic diseases (e.g., osteoporosis and bone metastases), but it will also facilitate the design of novel approaches for their precise treatment using drug or somatic gene therapy.